Distinct alpha-Synuclein species induced by seeding are selectively cleared by the Lysosome or the Proteasome in neuronally differentiated SH-SY5Y cells

J Neurochem. 2021 Mar;156(6):880-896. doi: 10.1111/jnc.15174. Epub 2020 Sep 22.

Abstract

A major pathological feature of Parkinson's disease (PD) is the aberrant accumulation of misfolded assemblies of alpha-synuclein (α-Syn). Protein clearance appears as a regulator of the 'α-Syn burden' underlying PD pathogenesis. The picture emerging is that a combination of pathways with complementary roles, including the Proteasome System and the Autophagy-Lysosome Pathway, contributes to the intracellular degradation of α-Syn. This study addresses the mechanisms governing the degradation of α-Syn species seeded by exogenous fibrils in neuronally differentiated SH-SY5Y neuroblastoma cells with inducible expression of α-Syn. Using human α-Syn recombinant fibrils (pre-formed fibrils, PFFs), seeding and aggregation of endogenous Proteinase K (PK)-resistant α-Syn species occurs within a time frame of 6 days, and is still prominent after 12 days of PFF addition. Clearance of α-Syn assemblies in this inducible model was enhanced after switching off α-Syn expression with doxycycline. Lysosomal inhibition led to accumulation of SDS-soluble α-Syn aggregates 6 days after PFF-addition or when switching off α-Syn expression. Additionally, the autophagic enhancer, rapamycin, induced the clearance of α-Syn aggregates 13 days post-PFF addition, indicating that autophagy is the major pathway for aggregated α-Syn clearance. SDS-soluble phosphorylated α-Syn at S129 was only apparent at 7 days of incubation with a higher amount of PFFs. Proteasomal inhibition resulted in further accumulation of SDS-soluble phosphorylated α-Syn at S129, with limited PK resistance. Our data suggest that in this inducible model autophagy is mainly responsible for the degradation of fibrillar α-Syn, whereas the proteasome system is responsible, at least in part, for the selective clearance of phosphorylated α-Syn oligomers.

Keywords: aggregation; alpha-synuclein; degradation; lysosome; phosphorylation; proteasome.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autophagy
  • Cell Line
  • Cell Survival
  • Doxycycline / pharmacology
  • Humans
  • Immunohistochemistry
  • Lysosomes / drug effects
  • Lysosomes / metabolism*
  • Neurons / metabolism*
  • Phosphorylation
  • Proteasome Endopeptidase Complex / drug effects
  • Proteasome Endopeptidase Complex / metabolism*
  • Signal Transduction / drug effects
  • Sirolimus / pharmacology
  • alpha-Synuclein / antagonists & inhibitors
  • alpha-Synuclein / genetics*
  • alpha-Synuclein / metabolism*

Substances

  • SNCA protein, human
  • alpha-Synuclein
  • Proteasome Endopeptidase Complex
  • Doxycycline
  • Sirolimus